US5776682AExpiredUtility
Male infertility y-deletion detection battery

Assignee: PROMEGA CORPPriority: Jun 7, 1995Filed: Sep 18, 1995Granted: Jul 7, 1998
Est. expiryJun 7, 2015(expired)· nominal 20-yr term from priority
Inventors:Marijo Kent First Alexander Agoulnik Ariege Muallem
Y10S435/81C12Q 2600/16C12Q 1/6883C12Q 1/6879C12Q 2600/156
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Claims
Abstract

The present disclosure describes a method for probing the integrity of a Y chromosome utilizing multiplex PCR reactions which amplify specific regions of the human Y chromosome which have been linked to normal fertility in human males. The method is capable of detecting deletion mutations within the Y chromosome which are predictive of human male infertility. A kit containing reagents needed to practice the method is also disclosed.
Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A method for detecting deletions in a Y chromosome which are indicative of male infertility comprising: (a) combining at least one plurality of distinct oligonucleotide primer pairs capable of priming at least one corresponding plurality of human X and Y chromosome loci selected from the group consisting of:   DYS209, DYF43S1, DYS210, DYS211, DYS33, DYS1, SMCX, DAZ(1);   DYS218, DYS219, DYS212, DYF53S1, DYS205, DYS281, MIC2;   DYS201, DYS241, DYS198, SRY, DYS197, DYS196, MIC2;   DYS240, DYS271, DYS221, KAL182, DAZ(2), MIC2;   DYS224, DYS226, DYS222, DYS227, MIC2;   DYF53S1, DYS229, DYZ1, DYS230, DAZ(3), DAZ(4), DAZ(5), MIC2;   SMCY, DYS217, DYS220, DYS223, DYS7, DYS237, DYS215, MIC2;   SMCY, DYS217, DYS220, DYS7, DYS237, DYS215, DAZ(6), MIC2;   DAZ(7), DAZ(8), DAZ(9), DAZ(10), DAZ(11), MIC2; and   YRRM1, SMCY, ZFY, BKM, SMCX;   with isolated genomic DNA of a test subject; then   (b) amplifying the at least one plurality of distinct oligonucleotide primer pairs by at least one corresponding multiplex polymerase chain reaction to yield locus-specific amplified chromosomal DNA fragments; then   (c) separating the amplified chromosomal DNA fragments; and then   (d) comparing the amplified chromosomal DNA fragments to corresponding amplified chromosomal DNA fragments from normal male subjects, whereby deletions in the Y chromosome of the test subject are detected.   
     
     
       2. The method according to claim 1, wherein in step (a) the isolated genomic DNA of the test subject is combined with at least one plurality of distinct oligonucleotide primer pairs selected from the group consisting of: SEQ. ID. NOS. 1-14, 95, and 96;   SEQ. ID. NOS. 15-26, 97, and 98;   SEQ. ID. NOS. 27-38, 97, and 98;   SEQ. ID. NOS. 39, 40, 43-48, 97-100;   SEQ. ID. NOS. 49-56, 97, and 98;   SEQ. ID. NOS. 59-62, 75, 76, 97, 98, and 115-122;   SEQ. ID. NOS. 71-74, 79-86, 97, 98, 101, and 102;   SEQ. ID. NOS. 71-74, 79, 80, 83-86, 97, 98, and 101-104;   SEQ. ID. NOS. 97, 98, and 105-114; and   SEQ. ID. NOS. 87-94, 13, and 14.   
     
     
       3. The method according to claim 2, wherein in step (a) the at least one plurality of distinct oligonucleotide primer pairs is combined with isolated genomic DNA of a human test subject. 
     
     
       4. The method according to claim 3, wherein in step (a) the at least one plurality of distinct oligonucleotide primer pairs is combined with isolated genomic DNA of a phenotypically male human test subject. 
     
     
       5. The method according to claim 3, wherein in step (a) the at least one plurality of distinct oligonucleotide primer pairs is combined with isolated genomic DNA of a human test subject of phenotypically ambiguous sexuality. 
     
     
       6. The method according to claim 3, wherein in step (a) the at least one plurality of distinct oligonucleotide primer pairs is combined with isolated genomic DNA of a phenotypically female human test subject. 
     
     
       7. The method according to claim 1, wherein in step (c), the amplified chromosomal DNA fragments are separated by gel electrophoresis. 
     
     
       8. The method according to claim 1, wherein in step (b) the at least one plurality of distinct oligonucleotide primer pairs amplified by at least one corresponding multiplex polymerase chain reaction are amplified by subjecting the at least one corresponding multiplex polymerase chain reaction to an initial denaturation at about 95° C. for about 2.5 minutes, and then cycling through 25 to 35 cycles of denaturation at about 95° C. for about 1 minute, annealing at about 61° C. for about 1 minute, and extension at about 72° C. for about 1 minute. 
     
     
       9. A method for detecting deletions in a Y chromosome which are indicative of male infertility comprising: (a) combining ten pluralities of distinct oligonucleotide primer pairs capable of priming ten corresponding pluralities of human X and Y chromosome loci selected from the group consisting of:   DYS209, DYF43S1, DYS210, DYS211, DYS33, DYS1, SMCX, DAZ(1);   DYS218, DYS219, DYS212, DYF53S1, DYS205, DYS281, MIC2;   DYS201, DYS241, DYS198, SRY, DYS197, DYS196, MIC2;   DYS240, DYS271, DYS221, KAL182, DAZ(2), MIC2;   DYS224, DYS226, DYS222, DYS227, MIC2;   DYF53S1, DYS229, DYZ1, DYS230, DAZ(3), DAZ(4), DAZ(5), MIC2;   SMCY, DYS217, DYS220, DYS223, DYS7, DYS237, DYS215, MIC2;   SMCY, DYS217, DYS220, DYS7, DYS237, DYS215, DAZ(6), MIC2;   DAZ(7), DAZ(8), DAZ(9), DAZ(10), DAZ(11), MIC2; and   YRRM1, SMCY, ZFY, BKM, SMCX;   with isolated genomic DNA of a test subject, wherein each of the ten pluralities of distinct oligonucleotide primer pairs with its respective test subject genomic DNA are disposed within separate receptacles; then   (b) amplifying the ten pluralities of distinct oligonucleotide primer pairs by ten corresponding multiplex polymerase chain reactions to yield locus-specific amplified chromosomal DNA fragments; then   (c) separating the amplified chromosomal DNA fragments; and then   (d) comparing the amplified chromosomal DNA fragments to corresponding amplified chromosomal DNA fragments from normal subjects, whereby deletions in the Y chromosome of the test subject are detected.   
     
     
       10. The method according to claim 9, wherein the isolated genomic DNA of the test subject is combined with the following ten pluralities of distinct oligonucleotide primer pairs: SEQ. ID. NOS. 1-14, 95, and 96;   SEQ. ID. NOS. 15-26, 97, and 98;   SEQ. ID. NOS. 27-38, 97, and 98;   SEQ. ID. NOS. 39, 40, 43-48, 97-100;   SEQ. ID. NOS. 49-56, 97, and 98;   SEQ. ID. NOS. 59-62, 75, 76, 97, 98, and 115-122;   SEQ. ID. NOS. 71-74, 79-86, 97, 98, 101, and 102;   SEQ. ID. NOS. 71-74, 79, 80, 83-86, 97, 98, and 101-104;   SEQ. ID. NOS. 97, 98, and 105-114; and   SEQ. ID. NOS. 87-94, 13, and 14.   
     
     
       11. The method according to claim 10, wherein in step (a) the ten pluralities of distinct oligonucleotide primer pairs are combined with isolated genomic DNA of a human test subject. 
     
     
       12. The method according to claim 11, wherein in step (a) the ten pluralities of distinct oligonucleotide primer pairs are combined with isolated genomic DNA of a phenotypically male human test subject. 
     
     
       13. The method according to claim 11, wherein in step (a) the ten pluralities of distinct oligonucleotide primer pairs are combined with isolated genomic DNA of a human test subject of phenotypically ambiguous sexuality. 
     
     
       14. The method according to claim 11, wherein in step (a) the ten pluralities of distinct oligonucleotide primer pairs are combined with isolated genomic DNA of a human male test subject with infertility due to azoospermia or oligospermia. 
     
     
       15. The method according to claim 11, wherein in step (a) the ten pluralities of distinct oligonucleotide primer pairs are combined with isolated genomic DNA of a phenotypically female human test subject. 
     
     
       16. The method according to claim 9, wherein in step (c), the amplified chromosomal DNA fragments are separated by gel electrophoresis. 
     
     
       17. The method according to claim 10, wherein in step (b) the ten pluralities of distinct primer pairs within the ten corresponding multiplex polymerase chain reactions are amplified by subjecting the ten corresponding multiplex polymerase chain reactions to an initial denaturation at about 95° C. for about 2.5 minutes, and then cycling through 25 to 35 cycles of denaturation at about 95° C. for about 1 minute, annealing at about 61° C. for about 1 minute, and extension at about 72° C. for about 1 minute. 
     
     
       18. A kit for detecting deletion mutations on a Y chromosome which are indicative of male infertility comprising: at least one first receptacle containing at least one corresponding plurality of locus-specific oligonucleotide primer pairs, said at least one corresponding plurality of oligonucleotide primer pairs capable of specifically priming at least one corresponding plurality of human X and Y chromosome loci selected from the group consisting of:   DYS209, DYF43S1, DYS210, DYS211, DYS33, DYS1, SMCX, DAZ(1);   DYS218, DYS219, DYS212, DYF53S1, DYS205, DYS281, MIC2;   DYS201, DYS241, DYS198, SRY, DYS197, DYS196, MIC2;   DYS240, DYS271, DYS221, KAL182, DAZ(2), MIC2;   DYS224, DYS226, DYS222, DYS227, MIC2;   DYF53S1, DYS229, DYZ1, DYS230, DAZ(3), DAZ(4), DAZ(5), MIC2;   SMCY, DYS217, DYS220, DYS223, DYS7, DYS237, DYS215, MIC2;   SMCY, DYS217, DYS220, DYS7, DYS237, DYS215, DAZ(6), MIC2;   DAZ(7), DAZ(8), DAZ(9), DAZ(10), DAZ(11), MIC2; and   YRRM1, SMCY, ZFY, BKM, SMCX;   at least one second receptacle containing at least one control DNA amplimer ladder corresponding to said at least one plurality of human X and Y chromosome loci; and   instructions for use.   
     
     
       19. The kit according to claim 18, wherein said at least one corresponding plurality of oligonucleotide primer pairs is selected from the group consisting of: SEQ. ID. NOS. 1-14, 95, and 96;   SEQ. ID. NOS. 15-26, 97, and 98;   SEQ. ID. NOS. 27-38, 97, and 98;   SEQ. ID. NOS. 39, 40, 43-48, 97-100;   SEQ. ID. NOS. 49-56, 97, and 98;   SEQ. ID. NOS. 59-62, 75, 76, 97, 98, and 115-122;   SEQ. ID. NOS. 71-74, 79-86, 97, 98, 101. and 102;   SEQ. ID. NOS. 71-74, 79, 80, 83-86, 97, 98, and 101-104;   SEQ. ID. NOS. 97, 98, and 105-114; and   SEQ. ID. NOS. 87-94, 13, and 14.   
     
     
       20. The kit according to claim 18, further comprising a supply of nanopure water, a supply of PCR-suitable buffer, a supply of PCR-suitable magnesium, and a supply of PCR-suitable dNTP&#39;s. 
     
     
       21. The kit according to claim 19, further comprising at least one third receptacle, wherein said supplies of nanopure water, PCR-suitable buffer, PCR-suitable magnesium, and PCR-suitable dNTP&#39;s are combined together at PCR-suitable concentrations and disposed therein. 
     
     
       22. The kit according to claim 18, wherein said at least one corresponding plurality of oligonucleotide primer pairs and said at least one control DNA amplimer ladder are contained respectively in said at least one first receptacle and said at least one second receptacle, in combination with PCR-suitable concentrations of nanopure water, PCR-suitable buffer, PCR-suitable magnesium, and PCR-suitable dNTP&#39;s. 
     
     
       23. The kit according to claim 18, further comprising a plurality of pre-sterilized microfuge receptacles. 
     
     
       24. The kit according to claim 18, further comprising a supply of Taq DNA polymerase. 
     
     
       25. The kit according to claim 18, further comprising a supply of sterile mineral oil. 
     
     
       26. The kit according to claim 18, further comprising a supply of molecular weight DNA control markers. 
     
     
       27. The kit according to claim 18, further comprising a supply of gel electrophoresis loading dye. 
     
     
       28. The kit according to claim 18, further comprising a supply of normal human female DNA, and a supply of normal human male DNA. 
     
     
       29. The kit according to claim 18, further comprising: ten first receptacles containing ten corresponding pluralities of oligonucleotide primer pairs capable of priming ten pluralities of human Y chromosome loci and non-Y linked control loci as follows:   DYS209, DYF43S1, DYS210, DYS211, DYS33, DYS1, SMCX, DAZ(1);   DYS218, DYS219, DYS212, DYF53S1, DYS205, DYS281, MIC2;   DYS201, DYS241, DYS198, SRY, DYS197, DYS196, MIC2;   DYS240, DYS271, DYS221, KAL182, DAZ(2), MIC2;   DYS224, DYS226, DYS222, DYS227, MIC2;   DYF53S1, DYS229, DYZ1, DYS230, DAZ(3), DAZ(4), DAZ(5), MIC2;   SMCY, DYS217, DYS220, DYS223, DYS7, DYS237, DYS215, MIC2;   SMCY, DYS217, DYS220, DYS7, DYS237, DYS215, DAZ(6), MIC2;   DAZ(7), DAZ(8), DAZ(9), DAZ(10), DAZ(11), MIC2; and   YRRM1, SMCY, ZFY, BKM, SMCX;   ten second receptacles containing ten control DNA amplimer ladders corresponding to said ten pluralities of human Y chromosome loci and non-Y linked control loci.   
     
     
       30. The kit according to claim 28, wherein said ten corresponding pluralities of oligonucleotide primer pairs are: SEQ. ID. NOS. 1-14, 95, and 96;   SEQ. ID. NOS. 15-26, 97, and 98;   SEQ. ID. NOS. 27-38, 97, and 98;   SEQ. ID. NOS. 39, 40, 43-48, 97-100;   SEQ. ID. NOS. 49-56, 97, and 98;   SEQ. ID. NOS. 59-62, 75, 76, 97, 98, and 115-122;   SEQ. ID. NOS. 71-74, 79-86, 97, 98, 101, and 102;   SEQ. ID. NOS. 71-74, 79, 80, 83-86, 97, 98, and 101-104;   SEQ. ID. NOS. 97, 98, and 105-114; and   SEQ. ID. NOS. 87-94, 13, and 14.   
     
     
       31. The kit according to claim 28, further comprising a supply of nanopure water, a supply of PCR-suitable buffer, a supply of PCR--suitable magnesium, and a supply of PCR-suitable dNTP&#39;S. 
     
     
       32. The kit according to claim 30, further comprising at least one third receptacle, wherein said supplies of nanopure water, PCR-suitable buffer, PCR-suitable magnesium, and PCR-suitable dNTP&#39;s are combined together at PCR-suitable concentrations and disposed therein. 
     
     
       33. The kit according to claim 28, wherein said ten corresponding pluralities of oligonucleotide primer pairs and said ten control DNA amplimer ladders are contained respectively in said ten first receptacles and said ten second receptacles, in combination with PCR-suitable concentrations of nanopure water, PCR-suitable buffer, PCR-suitable magnesium, and PCR-suitable dNTP&#39;s. 
     
     
       34. The kit according to claim 28, further comprising a plurality of pre-sterilized microfuge receptacles. 
     
     
       35. The kit according to claim 28, further comprising a supply of Taq DNA polymerase. 
     
     
       36. The kit according to claim 28, further comprising a supply of sterile mineral oil. 
     
     
       37. The kit according to claim 28, further comprising a supply of molecular weight DNA control markers. 
     
     
       38. The kit according to claim 28, further comprising a supply of gel electrophoresis loading dye. 
     
     
       39. The kit according to claim 28, further comprising a supply of normal human female DNA, and a supply of normal human male DNA. 
     
     
       40. A system for assessing the integrity of specific regions on the human Y chromosome, which regions are associated with human male infertility, comprising: at least one first receptacle containing at least one corresponding plurality of oligonucleotide primer pairs, said at least one corresponding plurality of oligonucleotide primer pairs capable of priming at least one corresponding plurality of human X and Y chromosome loci selected from the group consisting of:   DYS209, DYF43S1, DYS210, DYS211, DYS33, DYS1, SMCX, DAZ(1);   DYS218, DYS219, DYS212, DYF53S1, DYS205, DYS281, MIC2;   DYS201, DYS241, DYS198, SRY, DYS197, DYS196, MIC2;   DYS240, DYS271, DYS221, KAL182, DAZ(2), MIC2;   DYS224, DYS226, DYS222, DYS227, MIC2;   DYF53S1, DYS229, DYZ1, DYS230, DAZ(3), DAZ(4), DAZ(5), MIC2;   SMCY, DYS217, DYS220, DYS223, DYS7, DYS237, DYS215, MIC2;   SMCY, DYS217, DYS220, DYS7, DYS237, DYS215, DAZ(6), MIC2;   DAZ(7), DAZ(8), DAZ(9), DAZ(10), DAZ(11), MIC2; and   YRRM1, SMCY, ZFY, BKM, SMCX;   at least one second receptacle containing at least one control DNA amplimer ladder corresponding to said at least one plurality of human X and Y chromosome loci;   a third receptacle containing normal human female DNA;   a fourth receptacle containing normal human female DNA; and   instructions for use.
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